The long noncoding RNA LAL contributes to salinity tolerance by modulating LHCB1s’ expression in Medicago truncatula

Long non-coding RNAs (lncRNAs) are abundant in plants, however, their regulatory roles remain unclear in most biological processes, such as response in salinity stress which is harm to plant production. Here we show a lncRNA in Medicago truncatula identified from salt-treated Medicago truncatula is important for salinity tolerance. We name the lncRNA LAL, LncRNA ANTISENSE to M. truncatula LIGHT-HARVESTING CHLOROPHYLL A/B BINDING (MtLHCB) genes. LAL is an antisense to four consecutive MtLHCB genes on chromosome 6. In salt-treated M. truncatula, LAL is suppressed in an early stage but induced later; this pattern is opposite to that of the four MtLHCBs. The lal mutants show enhanced salinity tolerance, while overexpressing LAL disrupts this superior tolerance in the lal background, which indicates its regulatory role in salinity response. The regulatory role of LAL on MtLHCB1.4 is further verified by transient co-expression of LAL and MtLHCB1.4-GFP in tobacco leaves, in which the cleavage of MtLHCB1.4 and production of secondary interfering RNA is identified. This work demonstrates a lncRNA, LAL, functioning as a regulator that fine-tunes salinity tolerance via regulating MtLHCB1s’ expression in M. truncatula.


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Yang Zhao 1 , Yafei Liu 1 , Feiran Zhang 1 , Zeng-Yu Wang 2 , Kirankumar S. Mysore 3 , Jiangqi Wen 3 & Chuanen Zhou 1 Long non-coding RNAs (lncRNAs) are abundant in plants, however, their regulatory roles remain unclear in most biological processes, such as response in salinity stress which is harm to plant production.Here we show a lncRNA in Medicago truncatula identified from salt-treated Medicago truncatula is important for salinity tolerance.We name the lncRNA LAL, LncRNA ANTISENSE to M. truncatula LIGHT-HARVESTING CHLOROPHYLL A/B BINDING (MtLHCB) genes.LAL is an antisense to four consecutive MtLHCB genes on chromosome 6.In salt-treated M. truncatula, LAL is suppressed in an early stage but induced later; this pattern is opposite to that of the four MtLHCBs.The lal mutants show enhanced salinity tolerance, while overexpressing LAL disrupts this superior tolerance in the lal background, which indicates its regulatory role in salinity response.The regulatory role of LAL on MtLHCB1.4 is further verified by transient co-expression of LAL and MtLHCB1.4-GFP in tobacco leaves, in which the cleavage of MtLHCB1.4 and production of secondary interfering RNA is identified.This work demonstrates a lncRNA, LAL, functioning as a regulator that fine-tunes salinity tolerance via regulating MtLHCB1s' expression in M. truncatula.
Long noncoding RNA (lncRNA) are transcripts longer than 200 nucleotides that contain small reading frames or encode no peptides 1 .They are generated mostly by RNA polymerase II, while some by RNA polymerase I and RNA polymerase III 2 .According to their positions relative to coding genes, lncRNAs can be categorized into several classes: antisense of the coding genes, intronic, intergenic, upstream of the coding gene's promoter, promoter-associated and transcription start site-associated lncRNAs, et al 3 .
Recent studies have demonstrated their roles in transcriptional and posttranscriptional regulations 4 .Transcriptional regulation is very important for plants, because plants cannot move to avoid harm from the environment.Recently, more and more lncRNAs have been found to play roles in receiving signals from the outside and responding to different stresses, through proteins modifications and endo-siRNAs generation 5 .Therefore, lncRNAs play important roles, yet the one in salinity stress remains unclear.
In the past two decades, numerous studies have been conducted on plant salinity response, especially in the model plant Arabidopsis thaliana, and uncovered a number of key players involved in stress signaling and response 6 .In A. thaliana, the imposition of abiotic stresses leads to a transient burst of excess reactive oxygen species (ROS) production, and the disruption to ROS homeostasis has a negative effect on stress tolerance and on plant's growth 7,8 .On the first hand, the genes encoding the plant plasma membrane-localized nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidases, RESPIRATORY BURST OXIDASE HOMO-LOGUEs (RBOHs), are involved in ROS production when plants are imposed to stresses 9,10 .Production and accumulation of ROS, such as superoxide (O 2 zeathanxin epoxidase to catalyze zeathanxin to violaxanthin.Then violaxanthin is catalyzed into neoxanthin by neoxanthin synthase and neoxanthin to xanthoxin by 9-CIS-EPOXYCAROTENOID DIOXYGENASE (NCED).
In Arabidopsis, the chloroplast is important for ROS production 11 .In chloroplasts, photosystem II (PSII) outer antenna proteins consist of LHCBs.Among them, LHCB1, LHCB2 and LHCB3, form the major antenna complexes 12 .Electrons are produced from PSII and passed to photosystem I (PSI).Until now, the role of LHCB1s in salinity tolerance has not been elucidated clearly, neither the regulators of LHCB1s in salt stress.
Here in this work, we identified a lncRNA, LncRNA as an ANTISENSE for MtLHCB1s (LAL), from salt-treated Medicago plants, which was an antisense for four MtLHCB1 genes in a tandem.The Medicago lal mutants that disrupted the lncRNA's expression showed enhanced salinity tolerance accompanied with elevated MtLHCB1s' expression and activated ROS and ABA pathways.The transient expression assays of LAL with MtLHCB1.4 and knockdown of MtLHCB1s' plants diminishing lal's salinity tolerance, which showed that MtLHCB1s were targets of LAL.These findings demonstrate the important regulatory role of LAL in Medicago via modulate MtLHCB1s' expression under salinity tolerance.

Results
Upregulation of MtLHCB1s from salt-treated M. truncatula To study the mechanism underlying M. truncatula's response to salinity, we carried out a transcriptomic analysis of NaCl-treated plants.By comparing the mock-and NaCl-treated plants, we identified 4,412 differentially expressed genes (DEGs) with over two-fold changes in expression and false discovery rates (FDR) smaller than 0.001, including 1026 upregulated and 1814 downregulated DEGs at three hours post-treatment, and 1021 upregulated and 551 downregulated at 12 hours (Supplementary Data.1).Gene Ontology (GO) enrichment analysis revealed that genes encoding chloroplast-localized proteins were significantly enriched in the DEGs (q value < 0.01, Fig. 1a).Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed the similar pattern with the photosynthesis pathway significantly enriched in the DEGs (q value < 0.01, Fig. 1b).The most differentially regulated DEGs included 18 MtLHCBs involved in photosynthesis regulated at three hours and 12 hours with similar trends (Fig. 1c).
Phylogenetic analysis of LHCBs from M. truncatula and A. thaliana grouped 18 MtLHCBs into seven subgroups (Supplementary Fig. 1), in which MtLHCB1s showed the most induction in their expression.Among the five MtLHCB1s in the M. truncatula genome, MtLHCB1.1,MtLHCB1.2,MtLHCB1.3,MtLHCB1.4,and MtLHCB1.5 showed a similar induction in their expressions after NaCl treatment, and the former four genes are located on chromosome 6 as a tandem array (Fig. 1d).This result suggested the involvement MtLHCB1s in salinity tolerance in Medicago.
A long noncoding RNA, LAL, was identified antisense for MtLHCB1s From the alignment of RNA sequencing data, we noticed an antisense transcript overlapped with MtLHCB1 genes on chromosome 6 (Supplementary Fig. 2).We cloned the full length of this transcript and verified its ends using 5'-and 3'-RACE.Sanger sequencing revealed a 446-bp transcript consists of four exons, which was predicted to encode no protein by Coding Potential Calculator (CPC).Because the four exons of LAL were antisense for the tandem array of four MtLHCB1 genes, we named this gene as LONG NON-CODING RNA AS AN ANTISENSE FOR MtLHCB1s (LAL) (Fig. 2a).
Expression assay revealed that LAL displayed a ubiquitous expression pattern with higher transcription levels in leaf.The four MtLHCB1s were mainly expressed in leaf tissue, while MtLHCB1.5 was mostly expressed in flower, pod and root (Supplementary Fig. 3).We therefore focused on MtLHCB1.1-1.4 on chromosome 6.
In salinity-treated plants, LAL expression was suppressed at three hours but then activated more than ten times at six hours, followed by a gradual reduction to the basal level at 24 hours (Fig. 2b).In contrast, the MtLHCB1.1-1.4 exhibited an opposite pattern with more than ten-fold induction at three hours after salinity treatment and experienced a slow decline till 24 hours (Supplementary Fig. 4).As both LAL and MtLHCB1s responded to salinity treatment, we examined their response to ABA and H 2 O 2 .The result showed that the expression of LAL was suppressed and MtLHCB1s transcriptional levels induced under ABA or H 2 O 2 treatment (Fig. 2c, d and Supplementary Fig. 4).Our results identified a non-coding RNA, LAL, responding to salinities stress signals during salinity, ABA and H 2 O 2 treatment.

Disruption of LAL conferred enhanced salinity tolerance in lal-1
To study the role of LAL in salinity response, we screened M. truncatula Tnt1 mutant collection and found lal-1.lal-1 had an insertion in the LAL's first exon and also the MtLHCB1.4'scoding sequence, which disrupted both genes' functions (Fig. 3a, b).To assess the salinity tolerance in lal-1, we conducted a consecutive 4-week NaCl treatment and found that the mutant was tolerant to salinity with a high survival rate and stayed green till the end of the treatment (Fig. 3c, d).The indicator of oxidative damage, MDA, were assessed in lal-1 and wild-type (WT) plants, and the result showed a significantly lower contents of MDA in lal-1 compared with WT (Fig. 3e).The expressions of MtRboh genes involved the ROS synthesis pathway, including MtRbohB (Medtr3g098380), MtRbohD (Medtr3g098320) and a ROS scavenger gene, MtCAT1 (Medtr3g115370) were activated in lal-1 (Fig. 3f-h).As ABA plays a crucial role in salinity response, we further assessed the expression of genes involved in ABA synthesis.The genes in the ABA synthesis pathway, MtABA1 (Medtr5g017350), and MtNCED3 (Medtr2g070460) were significantly higher in lal-1 than those in the WT plants (Fig. 3i, j).
To verify the superior salinity tolerance in lal-1 was caused by its mutation, we overexpressed the LAL transcript in the homozygous lal-1 background.The lal-1;35 S:LAL#1, lal-1;35 S:LAL#2 and lal-1;35 S:LAL#3 transgenic lines restored the expression of LAL (Fig. 3b and Supplementary Fig. 5), with suppression of MtLHCB1.1,MtLHCB1.2 and MtLHCB1.3 compared with lal-1 but undetectable expression in MtLHCB1.4 (Fig. 3k-n).To assess the salinity tolerance in the transgenic plants, we conducted the same consecutive four-week NaCl treatment and found that the lal-1;35 S:LAL transgenic plants showed chlorosis and growth retardment like WT.The average survival rates of lal-1;35 S:LAL#1 and WT plants was 13% and 14%, respectively, which was significantly lower than 44% in lal-1 that stayed green by the end of NaCl treatment (Fig. 3c).MDA were assessed with comparable contents in WT and lal-1;35 S:LAL#1 (Fig. 3e).These results demonstrated that knockout of LAL conferred a superior tolerance to salt stress and overexpressing LAL diminished superior salinity tolerance in lal-1.
lal-1 showed enhanced salinity tolerance and had an insertion located both in LAL and MtLHCB1.4.To learn the role of MtLHCB1.4 in salinity tolerance, we identified a mtlhcb1.4mutant that carried one Tnt1 insertion in the CDS of MtLHCB1.4 located 179 bp before the transcriptional start site of LAL.mtlhcb1.4possessed an insertion in MtLHCB1.4,but not in LAL (Supplementary Fig. 6a).The expression result showed that the expression of MtLHCB1.4 was completely disrupted in mtlhcb1.4.We overexpressed MtLHCB1.4 in the WT background and the resulting transgenic plants were tested the expression of MtLHCB1.4 (Supplementary Fig. 6b).The fourweek-old WT, mtlhcb1.4 and transgenic plants were treated with a saline gradient NaCl to test their salinity tolerance.The mutant showed comparable salinity survival rate.Meanwhile, the overexpressing MtLHCB1.4 plants 35 S:MtLHCB1.4#1and #2 measured showed enhanced salinity tolerance (Supplementary Fig. 6c).The expressions of MtRboh genes and ROS scavenging MtCAT1 were elevated and the enzyme activity of MtCAT1 increased in transgenic plants (Supplementary Fig. 6d-g).The expressions of MtABA1 and MtNCED3 in ABA synthesis were increased in transgenic plants (Supplementary Fig. 6h, j).These results demonstrated the positive role of MtLHCB1.4 in salinity tolerance.

lal mutants displayed salinity tolerance with synthesis of ROS and ABA enhanced
To study the molecular function of LAL in salinity response, we screened our mutant collection and found another two mutants with Tnt1 insertions within the LAL gene, lal-1, lal-2 and lal-3.Both lal-2 and lal-3 had an insertion in the second intron of LAL, which corresponded to the intergenic region between MtLHCB1.2 and MtLHCB1.3 (Fig. 4a).In the homozygous lal-2 and lal-3 mutants, the LAL expression was undetectable while the expression levels of MtLHCB1s were significantly higher than those in WT, suggesting a negative regulation of MtLHCBs by LAL (Fig. 4b, c).
Salinity tolerance was assessed in the lal-2 and lal-3 mutants.The fourweek-old plants were treated with a saline gradient with 50 to 200 mM NaCl for four weeks.The WT plants showed severe chlorosis at three week posttreatment, while the lal mutants stayed green.At the end the experiment, the WT plants showed severe chlorosis and growth retardment, but the most mutant plants remained green.The average survival rates of lal-2 and lal-3 after the NaCl treatment were 49% and 41%, respectively, which were significantly higher than that of 15% in WT (Fig. 4d, e).Moreover, the MDA contents, the indicator of oxidative damage, in the lal mutants were significantly lower than that in WT (Fig. 4f).To understand the mechanism underlying salinity tolerance, we measured several hallmark responses to salt stress in the lal mutants.DAB staining detected higher ROS contents in lal-2 and lal-3 than that in the WT plants (Fig. 4g), accompanied with the elevated H 2 O 2 contents (Fig. 4h) and the expressions of MtRboh genes involved the ROS pathway, including MtRbohB, MtRbohD, (Fig. 4i, j).The expression of MtCAT1, was significantly higher in lal-2 and lal-3 than WT (Fig. 4k), and its activity also showed a two-fold increase in the mutant leaves (Fig. 4l).These results showed that the ROS synthesis and scavenging pathways were constitutively activated in the lal mutants.MtABA1, and MtNCED3 were significantly higher in lal-2 and lal-3 mutants than those in the WT plants (Fig. 4m, n).We also measured the ABA contents, and the result showed that ABA accumulated to higher levels in lal mutants compared to the WT plants (Fig. 4o).These results demonstrated ABA biosynthesis pathway was activated in lal mutants.
For MtLHCB1s are PSII outer antenna proteins, we investigated the impact of LAL on the photosynthetic apparatus by chlorophyll fluorescence parameters such as the maximal quantum yield of photosystem II (Fv/Fm), the photochemical yields of photosystem II (YII), non-photochemical quenching (NPQ) and photochemical quenching (qL).The four-week-old WT, lal-2 and lal-3 mutant plants were treated with 100 mM NaCl for three days.The decrease of Fv/ Fm was because of the impaired photochemical activity in salt treatment.The WT and mutant plants showed comparable values of Fv/Fm (Supplementary Fig. 7a).YII reflected the impairment to photosystem II caused by salinity showing the similar trends with Fv/ Fm in WT and mutant plants (Supplementary Fig. 7b).Interestingly, NPQ, transferring photons as heat to protect light-harvesting complexes, showed a remarkable increase in lal-2 and lal-3 mutants (Fig. 4p).In photosynthesis, the efficiency of light energy transferred to photochemistry, qL was remaining the high values in lal-2 and lal-3 (Fig. 4q).Hence, the photosynthesis of lal showed enhanced photoprotection under salinity stress.

LAL performed salinity tolerance via regulating MtLHCB1s' expression
To further study the relationship between LAL and MtLHCB1, the transient expression assay of LAL and MtLHCB1.4-GFP was investigated in tobacco leaves (Fig. 5a).The results showed the GFP signal intensity of MtLHCB1.4 transferred with LAL was diminished in transfection with 35 S:LAL compared with that without LAL (Fig. 5b).The suppression of MtLHCB1.4-GFPexpression was verified by qRT-PCR (Fig. 5c and Supplementary Fig. 8).These results indicate a suppression role of LAL on MtLHCB1.4.
To further determine if LAL cause the post-transcriptional regulation of MtLHCB1s, we carried out 5'-RACE in the tobacco leaves co-expressing MtLHCB1.4 and LAL and found the cleavage products of the MtLHCB1.4.In the tobacco leaves co-expressing MtLHCB1.4 and LAL, we detected a siRNA, siRNALAL corresponding to the MtLHCB1.4 cleavage site (Fig. 5e).The abundance of siRNALAL was also found in a lesser extent in lal-2 compared with WT (Fig. 5f).Due to the high identities among these MtLHCB1s, we constructed MtLHCB1s-RNAi lines in lal-2 backgrounds and a total of sixteen transgenic lines were generated.The qRT-PCR results showed that MtLHCB1.1,MtLHCB1.2,MtLHCB1.3 and MtLHCB1.4 were statistical significantly suppressed (Fig. 5j).The salinity tolerance was assessed in these RNAi lines along with lal-2.The results showed the salinity tolerance observed in lal-2 were diminished in lal-2;RNAi-MtLHCB1s#1 and lal-2;RNAi-MtLHCB1s#2 with the survival rates about 18% and 21%, statistical significantly lower than that of lal-2, about 48% (Fig. 5k).Hence, the salinity tolerance of lal mutant relied on MtLHCB1s functions.

Discussion
Here we identified a lncRNA in M. truncatula, LAL, an antisense to four consecutive MtLHCB genes on chromosome 6.lal-2 and lal-3 mutants showed enhanced salinity tolerance accompanied with elevated MtLHCB1s' expression and activated ROS and ABA pathways.The knockdown of MtLHCB1s' plants diminished lal's salinity tolerance, showing that MtLHCB1s were targets of LAL.
LHCB1s encode apoproteins of photosystem II and are important for plants.But, the role of LHCB1 in salinity tolerance was unclear.It is reported previously that in Arabidopsis lhcb1 was insensitive to ABA 13,14 , and ABA activated the expressions of LHCBs in low concentration, and suppressed their expressions in high concentration, which accumulated in the process of salinity stress.It was shown in our study that overexpressing MtLHCB1.4 enhanced salinity tolerance.In overexpressing MtLHCB1.4 plants, the ROS synthesis and scavenging pathways were both activated, while the biosynthesis pathway of ABA was enhanced (Supplementary Fig. 6).Our findings help to understand the positive roles of MtLHCB1s protein in plant salinity tolerance and ROS homeostasis.
In Arabidopsis, LHCB1 has 5 homologs, LHCB1.1,LHCB1.2,LHCB1.3,LHCB1.4 and LHCB1.5, among which three are located on chromosome 1, two on chromosome 2 15 .Take MtLHCB1.4,for example, MtLHCB1.4 is the homolog of LHCB1s, with identities higher than 80% and the homolog of MtLHCB1s, with identities higher than 93%.Interestingly, salinity-responsive MtLHCB1s form a cluster showing a tandem pattern in genome.The tandem-located targets with high identities were also found in MtCBFs in cold tolerance 16 .In our study, four MtLHCB1s shared similar expression pattern (Supplementary Fig. 3) and similar stress responses to LAL (Fig. 2 and Supplementary Fig. 4).Hence LAL is the antisense of these tandem-located MtLHCB1s, this LAL-MtLHCB1s module makes multiple MtLHCB1s salinity-responsive simultaneously.
In this work, when MtLHCB1.4 was co-expressed with LAL, the signals of MtLHCB1.4 were diminished, which suggested the sense-antisense regulation between LAL and MtLHCB1.4 (Fig. 5).In mammals, every cell of female embryo in early developmental stages only keeps one functional X chromosome from the two from parents and silences the other one 17 .The lncRNAs Xist initiates the silencing and the antisense Tsix blocks it on the active X.These two partners, Xist and Tsix, form complementary duplexes processed by Dicer into small RNAs 18 .In our work, siRNALAL complemented to MtLHCB1s and its expression was diminished in lal-2 mutant (Fig. 5).The disruption of complementation between LAL and MtLHCB1s probably led to decrease of siRNAs and upregulation of MtLHCB1s.Interestingly, in lal-1;35 S:LAL#1, the expressions of MtLHCB1.1,MtLHCB1.2, and MtLHCB1.3 were reduced and the salinity tolerance vanished (Fig. 3).Via the cluster-assembling targeting MtLHCB1s homologs, LAL performed salinity tolerance in a dosage-depending manner.This finding helps to guide agricultural engineering in alfafa.

Plant materials and growth conditions
The plant materials used in this work were in the M. truncatula ecotype R108 background.Mutants were requested from the Noble Research Institute Tnt1 Mutant collection 19 .The lal-1/NF20242, lal-2/NF19927, lal-3/ l The CAT activity in WT, lal-2 and lal-3.m The expression level of MtABA1 in WT, lal-2 and lal-3 by qRT-PCR.n The expression level of MtNCED3 in WT, lal-2 and lal-3 by qRT-PCR.o The ABA contents of WT, lal-2 and lal-3 mutants.Error bars represent the SD from three biological replicates (grey dots).Columns labeled with asterisks indicate significant differences from those in WT (*P < 0.05, student's t test).n = 3. MtUBIQUITIN was used as the internal control in qRT-PCR.The expression level in WT was set at 1.0.Response to salinity stress on photosynthetic parameters NPQ (p) and qL (q) of the WT, lal-2 and lal-3 mutants at different time points of NaCl-treatment.Values are shown by mean ± SD (grey dots with black circles).Columns labeled with asterisks indicate significant differences from those in WT (*P < 0.05, student's t test).n = 25.NF17218, and mtlhcb1.4/NF14097mutants were identified using genespecific and Tnt1-specific primers (listed in Supplementary Table 1).Seeds were scarified mechanically and vernalized at 4 °C on moist filter paper for one week before transfer to soil.Plants were grown in a growth chamber at 22 °C under a 16-h-light/8-h-dark cycle with 150 μmol/m 2 /s light intensity and 70-80% relative humidity.To determine gene expression in different tissues, root, stem, leaf, flower and shoot tip were harvested from four-weekold plants.Pods were harvested from eight-week-old plants.For each biological replicate, 25 plants were used.Three biological replicates were included.

Salinity treatment
For stress treatments, four-week-old plants were treated with 100 mM NaCl, 100 µM ABA or 1 mM H 2 O 2 .Leaf samples were harvested at different time points after treatment for RNA extraction.Salinity tolerance was scored after a four-week NaCl treatment starting with one-week treatment with 50 mM NaCl and an increment of 50 mM per week till 200 mM 20 .For each biological replicate, 25 plants were used.Three biological replicates were included.

RNA isolation and quantitative real-time PCR (qRT-PCR)
Harvested tissue samples were frozen immediately in liquid nitrogen, from which RNA was extracted using RNeasy Mini Kit (Qiagen, U.S.A.) and treated with DNase I. RNA quantity was measured on a NanoDrop 2000 Spectrophotometer (NanoDrop Technologies, U.S.A).One µg of total RNA was transcribed into cDNA using ThermoScript RT-PCR system (Ther-moFisher, U.S.A), and qRT-PCR was performed on a CFX Connect Real-Time PCR Detection system (Bio-Rad, U.S.A) using SYBR Green PCR Master Mix (Roche, Switzerland) in three biological replicates.A MtUBI-QUITIN gene, Medtr3g110110, and a tobacco gene L25, Genbank:L18908.1,were used as internal controls 21 .siRNA cDNA was transcribed using miRcute Plus miRNA First-Strand cDNA kit (TIANGEN, China), and qRT-PCR was performed using miRcute Plus miRNA qPCR kit (TIAN-GEN, China) in three biological replicates, with 25 plants in each replicate.And MtU6 gene, was used as an internal control 22 .Relative gene expression was calculated according to the ΔΔC T method.The primer sequences used for qRT-PCR were designed using the Primer Express 3.0 software and listed in Supplementary Table 1.

Transcriptomic analysis
For the transcriptomic analysis, four-week-old plants were treated with water or 100-mM NaCl, and the leaf samples were harvested at three hours and 12 hours after treatment from mock-and NaCl-treated plants.Three biological replicates were included, with 25 plants in each replicate.RNA was extracted from the leaf samples as described above.RNA libraries were constructed and sequenced on a BGISEQ-500 platform following the manufacturer's instructions (BGI Genomics, China).Raw reads were filtered by SOAPnuke (v1.5.2; https://github.com/BGI-flexlab/SOAPnuke),and then aligned to the M. truncatula reference transcriptome (version 4.0) by Bowtie2 (v2.2.5) using the default parameters.Read counts were calculated using RSEM 23 , and differentially expressed genes (fold change >= 2 and false discovery rate (FDR) < 0.001) were called using the R package DEGseq.Enrichment of Gene Ontology (GO) terms and KEGG pathways were assessed using a hypergeometric test and the p values were adjusted by the FDR method [24][25][26] .CPC2 was used for the CPC analysis 27 .

Plasmid construction and plant transformation
The full length of LAL(Genbank accession number OR463063) transcript was confirmed by 5' and 3' rapid-amplification of cDNA ends (RACE) using SMARTer RACE 5'/3' kit (Takara, Japan) 28 .The gene-specific primers (listed in Supplementary Table 1) were designed based on the transcript sequence and used to clone LAL into a pENTR-TOPO vector.The resulting pENTR-LAL plasmid was recombined with a destination vector pEarley-Gate100 or pCAMBIA3301 to produce construct 35 S:LAL.The coding sequence (CDS) of MtLHCB1.4 was amplified from a cDNA sample prepared from leaf RNA with c-MtLHCB1.4-forwardand reverse primers and then used to generate pENTR-MtLHCB1.4,35 S:MtLHCB1.4and 35 S:MtLHCB1.4-GFPconstructs (listed in Supplementary Table 1) with destination vectors pEarleyGate100 and pEarleyGate103 29 .A 220-bp exon of MtLHCB1.4 was cloned into pH7GW1WG2 to generate the 35 S:MtLHCB1s-RNAi construct.
For M. truncatula transformation, target constructs were introduced into an Agrobacterium strain EHA105 and then used to transform Medicago leaf disc 30 .The transgenic plants were genotyped with a 35 S primer and a gene-specific primer (listed in Supplementary Table 1).The positive transformants were accessed for the target gene expression by qRT-PCR.Columns labeled with asterisks indicate significant differences from those in WT (*P < 0.05, student's t test).Complementary sequences of MtLHCB1.1 (g), MtLHCB1.2(h) and MtLHCB1.3 (i) to siRNALAL.j The expression level of MtLHCB1.1,MtLHCB1.2,MtLHCB1.3 and MtLHCB1.4 in lal-2, lal-2;RNAi-MtLHCB1s#1 and lal-2;RNAi-MtLHCB1s#2 plants by qRT-PCR.MtUBIQUITIN was used as the internal control.The expression level in lal-2 was set at 1.0.Error bars represent the SD from three biological replicates (grey dots).Columns labeled with asterisks indicate significant differences from those in lal-2 (*P < 0.05, student's t test).k The plant survival rates measured after the four-week treatment of salinity.Error bars represent the SD from three biological replicates (grey dots).Columns labeled with asterisks indicate significant differences from those in lal-2 (*P < 0.05, student's t test).l Four-week-old plants of lal-2, lal-2; RNAi-MtLHCB1s #1 and lal-2;RNAi-MtLHCB1s#2 plants after a four-week successive exposure to 50, 100, 150 and 200 mM NaCl.Bar = 5 cm.n = 3.

Transient expression assays
OD 600 value of 0.4) per plant and incubated in the dark at 25 °C for 24 hours.Leaves with comparable BAR expression were observed at 488-nm wavelength for the GFP fluorescence using a Zeiss LSM900 confocal microscopy (Zeiss, Germany).At least five images were taken under identical conditions and the relative intensity of GFP was assessed using the ImageJ software.The RNA of leaves co-expressing LAL and MtLHCB1.4 were extracted and the RLM 5'-RACE (RNA ligase mediated rapid amplification of 5' cDNA end) were performed with FirstChoice RLM-RACE kit (Invitrogen, U.S.A) 22 .Three biological replicates were included, with 25 plants in each replicate.

Statistics and reproducibility
Data processing and statistical analysis were performed using Microsoft Excel 2010.A student's t-test was used to evaluate the significance of the differences between two samples 31 .Three biological replicates were included, with 25 plants in each replicate.
Determination of peroxide level, catalase activity and ABA content H 2 O 2 levels were visualized by DAB staining of leaf samples from fourweek-old plants.H 2 O 2 levels and catalase activities were measured with commercial kits purchased from Beyotime Institute of Biotechnology (Haimen, China).ABA contents of leaf samples from four-week-old plants were assessed by LC-MS-MS 7 .Three biological replicates were included, with 25 plants in each replicate.
Chlorophyll fluorescence measurements Measurements were carried out using a PAM-2500 (Heinz Walz GmbH, Germany) pulse amplitude modulation fluorometer.The plants were darkadapted before the measurements and photosynthetic parameters were calculated by the PamWin software with provided automated induction and light curve routine 15 .Three biological replicates were included, with 25 plants in each replicate.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Fig. 1 |
Fig. 1 | Enrichment analysis of M. truncatula WT plants after NaCl treatment.a The GO enrichment analysis of DEGs at three hours after NaCl treatment.b The KEGG enrichment analysis of DEGs at three hours after NaCl treatment.c The heatmap of MtLHCBs and the homologs at three hours and 12 hours after NaCl treatment.The qRT validation of MtLHCB1.1,MtLHCB1.2,MtLHCB1.3,MtLHCB1.4,MtLHCB1.5 and MtLHCB1.6(d) at three hours after NaCl treatment.MtUBIQUITIN was used as the internal control.The expression level at 0 hour was set at 1.0.Error bars represent the SD from three biological replicates (grey dots).Columns labeled with asterisks indicate significant differences from those in WT (*P < 0.05, student's t test).n = 3.

Fig. 2 |
Fig. 2 | LAL's expression under salinity, ABA and H 2 O 2 treatment.a The scheme of LAL and MtLHCB1.1,MtLHCB1.2,MtLHCB1.3 and MtLHCB1.4 genes structure.Empty boxes represent MtLHCB1s, and black boxes represent LAL.Bar = 400 bp.b The expression level of LAL at different time points by qRT-PCR in M. truncatula WT plants under salinity treatment.c The expression level of LAL at different time points by qRT-PCR in M. truncatula WT plants under ABA treatment.d The expression level of LAL at different time points by qRT-PCR in M. truncatula WT plants under H 2 O 2 treatment.MtUBIQUITIN was used as the internal control.The expression level in 0 hour was set at 1.0.Error bars represent the SD from three biological replicates (grey dots).Columns labeled with asterisks indicate significant differences from those in 0 hour (*P < 0.05, student's t test).n = 3.

Fig. 4 |
Fig. 4 | lal showed salinity tolerance with enhanced ROS and ABA synthesis pathways.a The scheme of LAL and MtLHCB1.1,MtLHCB1.2,MtLHCB1.3 and MtLHCB1.4 genes structure and Tnt1 insertions in LAL of lal-1, lal-2 and lal-3.Empty boxes represent MtLHCB1s, and black boxes represent LAL.Three arrows indicate the positions where Tnt1 inserts in LAL.Bar = 400 bp.The expression level of LAL (b) and MtLHCB1s (c) in lal-2 and lal-3 by qRT-PCR.d Four-week-old plants of WT, lal-2 and lal-3 mutants after a four-week successive exposure to 50, 100, 150 and 200 mM NaCl.Bar = 5 cm.e The plant survival rates of WT, lal-2 and lal-3 measured after the four-week treatment of salinity.f The MDA content of WT, lal-2 and lal-3 mutants.g The DAB staining of WT, lal-2 and lal-3 mutants.Bar = 1 cm.h The H 2 O 2 contents of WT, lal-2 and lal-3 mutants.The expression level of MtRbohB (i), MtRbohD (j) and MtCAT1 (k) in WT, lal-2 and lal-3 by qRT-PCR.